The temporal and spatial patterns of DNA replication are fundamental aspects of genome biology. They correlate with patterns of transcriptional regulation, chromatin modification, nuclear structure and genome evolution. Furthermore, replication timing changes as cells differentiate, and disruption of replication timing correlates with genome instability, suggesting an intimate relation between replication timing and other important aspects of genome metabolism. However, the limitations of current techniques for assaying replication kinetics are limiting progress in the field. Genomic approaches to mapping replication kinetics suffer from low resolution and low sensitivity, preventing the identification f individual replication origins. Single locus techniques are laborious, restricting the number of experiments than can feasibly be done. We propose to develop an efficient, high-throughput, single-molecule, genome-wide replication mapping technology that we call optical replication mapping. This approach will combine in vivo labeling of replicated DNA with state-of-the-art optical mapping of megabase-sized single DNA molecules, allowing us to visualize patterns of DNA replication on tens of thousands of individual chromosomal fragments covering the genome to a thirty-fold depth. Successful development of optical replication technology will allow us and other groups to answer fundamental questions about DNA replication kinetics. Furthermore, it will provide accurate information about replication timing for workers in many other fields, essential to understand how replication timing influences other critical aspects of genome metabolism.